Protein Expression and Purification

Wild type CynD and variants were cloned into the expression vector pET28a using NdeI and XhoI restriction sites and transformed into MB1837 (BL21 pLysS) cells. Protein was expressed as described in the previous section and the pellet was stored at -20°C overnight. Cells were thawed and lysed at room temperature with B-PER II Protein Extraction Reagent (Thermo Scientific) using 2 mL of B-PER II reagent per gram of cell pellet supplemented with 0.5 mg/ml lysozyme, 10 μg/ml DNase and 1X EDTA-free protease inhibitors as recommended by the manufacturer. The cell lysate was centrifuged at 13,000 rpm for 15 min at 4°C. Purification of hexahistidine-tagged proteins was performed at 4°C using the HisPur Cobalt Purification Kit (Thermo Scientific). Desalting of the purified protein at 4°C used Zeba Spin Desalting Columns (Thermo Scientific) and the protein was resuspended in 0.1 M MOPS pH 7.7. Protein concentration was determined using the NanoDrop ND-1000 at 280nm using the predicted his-tagged protein extinction coefficient of 58,790 cm^-1 M^-1 and molecular weight of 39,654 g/mol. SDS-PAGE confirmed the purity of the protein. Purified proteins were stored in aliquots at -80°C.